This invention relates to methods and formulations for mediating virus attachment and infection, and more particularly relates to methods and formulations for mediating adeno-associated virus attachment and infection.
Adeno-associated virus (AAV) is a human parvovirus that infects a broad range of cell types including human, non-human primate, canine, murine, and avian. A member of the Parvoviridae family, AAV is a small non-enveloped single-stranded DNA virus of 20-25 nm which has an unique requirement for a helper virus (e.g., adenovirus or herpes simplex virus) to complete its lytic cycle (R. W. Atchison et al., (1965) Science 149:754; M. D. Hoggan et al, ((1966) Proc. Natl. Acad. Sci. USA 55:1457; J. L. Melnick et al., (1965) J. Bacteriol. 90:271). In the absence of helper virus, AAV still infects the target cell, but integrates into the host genome and establishes latency. Unique among eukaryotic DNA viruses, the AAV genome can integrate site specifically into human chromosome 19 (R. M. Kotin et al., (1990) Proc. Natl. Acad. Sci. USA 87:2211; R. J. Samulski et al., (1991) EMBO J. 10:3941; R. J. Samulski, (1993) Curr. Opin. Genet. Dev. 3:74; C. Giraud et al., (1994) Proc. Natl. Acad. Sci. USA 91:10039; C. Giraud et al., (1995) J. Virol. 69:6917). This property has drawn considerable attention to the potential use of AAV as a gene therapy vector, although little is known about the initial events of AAV infection (R. J. Samulski, (1995) Adeno-associated virus-based vectors for human gene therapy, p. 232-271. In K. M. Hui (ed.), Gene therapy: from laboratory to the clinic. World Scientific Publishing Co., Singapore, Singapore; C. McKeon et al., (1996) Hum. Gene Ther. 7:1615; D. M. McCarty et al, (1997) Adeno-associated viral vectors, p. 62-78. In M. Strauss and J. Barranger (ed.), Concepts in gene therapy. Walter de Gruyter, Bellin. N.Y.; R. J. Samulski, (1997) Development of adeno-associated virus as a vector for in vivo gene therapy, p. 197-203. In L. M. Houdebine (ed.), Transgenic animals: generation and use. Harwood Academic Publishers, Chur, Switzerland). In particular, the recombinant AAV or rAAV vector system is well characterized and is the subject of increasing development as a vector for gene delivery (see, C. McKeon et al. (1996) Hum. Gen. Ther. 7:1615). In general, AAV vectors are generated by deleting rep and cap genes and replacing them with genes intended for delivery into the cell. Additionally, producer cells that contain rep and cap may be used to package the gene therapy vectors into the AAV capsid particle (B. J. Carter, (1996) Nature Biotechnology 14:1725).
Despite this growing interest in AAV, the events that govern the initial AAV infection remain poorly understood. The primary event of any viral infection is attachment of virus to the host cell. A wide variety of cell surface molecules are now known to serve as viral attachment receptors. However, the mechanism by which AAV attaches to its host cell has heretofore not been delineated. AAV has a very broad host range and infects a wide variety of cell types, suggesting that the virus uses a ubiquitous receptor to mediate infection. Identification of the initial virus-host cell interactions necessary for efficient AAV infection is not only important for the general understanding of parvovirus infection, but also for the effective use of AAV as a gene therapy vector.
Although the initial events in the life cycle of AAV are not well understood, previous studies suggest that AAV infects cells through interaction with a specific host cellular receptor (H. Mizukami et al., (1996) Virology 217:124; S. Ponnazhagan et al., (1996) J. Gen. Virol. 77:1111). AAV appears to exhibit saturation binding to HeLa cells. In addition, cellular attachment of AAV is sensitive to trypsin treatment, suggesting a protein component is responsible for binding. Id
The lack of knowledge concerning the receptor of AAV has introduced significant obstacles to the development of reliable techniques for both isolating and using AAV as a means for gene therapy. For example, purification of AAV is generally conducted using techniques that ultimately involve the use of a CsCl gradient. There are certain disadvantages in using these techniques, primarily because CsCl is toxic and thus requires special handling. It would be highly desirable to develop a milder and less dangerous means of isolating AAV viral particles.
An additional obstacle to the use of AAV as a reliable gene therapy vector has been the difficulty in infecting certain types of cells with the vector. Experiments in cultured cells have shown that AAV vectors are efficient for delivery of genes to both dividing and non-dividing cells. However, these experiments have also shown that the efficiency and both expression and metabolic activation may vary with the cell type and the physiological state of the cell (C. McKeon et al., (1996) Hum. Gen. Ther. 7:1615). In particular, progenitor or stem cells (e.g., bone marrow CD34+ cells) have been found to be difficult to infect with the AAV vector. Additionally, in some cell types, persistence and expression of a heterologous gene carried by the vector are not well maintained. Finally, even when it is known that certain cell types are generally permissive to infection by AAV, is appears that there is diversity among individual cell donors as to whether or not any particular donor""s cells will permit infection by the AAV vector. It would be highly desirable to have means for the effective infection of stem cells and rare cell types, as well as the means for introducing the AAV vector into cells that may not naturally express the AAV receptor, or may not naturally produce the molecular substituents necessary for the attachment and internalization of the virus.
Accordingly, there is a need in the art for improved methods and reagents for purifying AAV and rAAV vectors. In addition, there is a need in the art for methods of modifying the wild-type tropism of AAV vectors for use in gene therapy and for screening cells for permissiveness to transduction by AAV vectors.
The methods, AAV vectors, and formulations of the present invention are based on the surprising discovery that has identified cell surface heparin and heparan sulfate (HS) proteoglycan as the primary cellular receptors for AAV. It has also been discovered that AAV interacts specifically with cell surface heparin and heparan sulfate glycosaminoglycans (GAG), and not other glycosaminoglycans. Further, it has now been determined that the presence of HS GAG on the cell surface directly correlates with the efficiency by which AAV can infect cells.
Moreover, a role has been established for xcex1vxcex25 integrin in AAV infection. AAV virions physically interact with the xcex25 subunit of xcex1vxcex25 integrin. Using genetically defined cell lines that either lack or express xcex1vxcex25, it has been demonstrated that cell surface expression of this integrin promotes AAV infection. The present investigations suggest that xcex1vxcex25 integrin acts to facilitate the internalization of AAV bound to cell surface heparin and HS proteoglycans into the cell. This is the first report of the involvement of an integrin in a parvovirus infection.
These discoveries have led to the development of methods and formulations that mediate the infection of a broad range of cell types with AAV, including cells that are typically non-permissive for infection by AAV. Additionally, these discoveries have led to the development of methods of purifying AAV using receptor-like molecules that bind to AAV, and methods of screening cell samples for their permissiveness to infection with AAV. Furthermore, these discoveries have elucidated new strategies for modifying the natural tropism of AAV, in particular, for use in gene therapy.
Accordingly, a first aspect of the present invention is a method of facilitating attachment of AAV to a cell, and infection of a cell by AAV, by contacting the cell with a soluble artificial receptor or soluble receptor-like molecule that mediates attachment and infection of AAV into the cell. This aspect of the invention is based on the observation that low concentrations of soluble heparin, HS and high molecular weight dextran sulfate enhance AAV infection. Heparin, HS, and other polyanionic molecules are known to attach to the cell surface. Therefore, exogenous heparin, HS, GAGs and other polyanionic molecules (preferably, heparin and HS) can mediate AAV attachment to and infection of cells that do not typically express heparin or HS on the cell surface (or that express these molecules at low concentrations).
The discovery that heparin and HS proteoglycans are the receptor for AAV has also led to the development of a further aspect of the present invention, which is a method of purifying and/or concentrating AAV. According to one embodiment, a receptor-like molecule is immobilized to a matrix to form a solid support that binds the AAV. Sarnples suspected of containing AAV are then contacted with the immobilized receptor-like molecules. The bound AAV is eluted (e.g., with a high salt wash) and collected. This method may be practiced in numerous alternative embodiments, for example, by affinity chromatography, by batch purification methods (e.g., with magnetized beads), or by immobilizing the receptor-like molecule to a polymeric surface such as a plate or a tube. As a further alternative, the matrix can be a material such as fiberglass, cellulose acetate, nitrocellulose, or nylon. Such matrices can be advantageously employed to bind AAV, e.g., for titering or purification for analytical purposes.
The receptor of AAV having been determined relates to the a further aspect of the present invention, which is a method of facilitating or enhancing attachment of AAV to a cell, thus increasing the efficiency of AAV infection into a cell. In one particular embodiment of this method, the AAV capsid is mutated using techniques known to those skilled in the art, such that the mutant AAV exhibits enhanced attachment to cellular receptors and thus may increase infectivity of the AAV into the cell. More particularly, at least one of the AAV binding sites for heparin/HS is mutated, such that binding is enhanced. According to another embodiment, binding of AAV to a cell is facilitated or enhanced by upregulating the expression of receptors (e.g., heparin or HS) on the surface of the cell. Exemplary compounds that upregulate cell surface expression of heparin and HS are transforming growth factored, sodium butyrate, and fibroblast growth factor.
A further aspect of the present invention is a method of inhibiting or preventing binding of AAV to a cell. In one embodiment of this method, the AAV is mutated using techniques known to one skilled in the art, such that binding of AAV is prevented or inhibited. In particular, at least one of the AAV binding sites for heparin and/or HS is mutated (e.g, by deletion or by replacing basic amino acids with neutral amino acids) such that binding of AAV to cell surface receptors is prevented or inhibited. In another embodiment of this method, a cell that naturally expresses the AAV receptor is treated with an enzyme or reagent that removes or alters the natural AAV receptor, such that AAV binding to the cell is prevented or reduced or the AAV receptor can no longer mediate infection of the cell by AAV. In yet another embodiment of this method, AAV virus is treated with molecules (e.g., heparin, HS, high molecular weight dextran sulfate, antibodies) that have been determined to block the interaction between AAV and the AAV receptor at concentrations effective to inhibit or prevent binding of AAV to the cell, compared to that which would occur in the absence of such treatment.
A further aspect of the present invention is a method of screening a cell for permissiveness to AAV infection by detecting the presence or absence, or alternatively, the abundance, of the AAV receptor on the cell surface. In this method, a cell or sample of cells is contacted with, for example, an antibody to the AAV receptor. Binding of the receptor to the antibody is then detected and visualized by techniques that are readily available to one skilled in the art. This method finds particular use in screening potential donors for cells that may be used in gene therapy, in screening recipients for permissiveness to gene therapy using an AAV vector, and in screening cells for potential use as producer cells for the AAV vector.
A further aspect of the present invention are formulations containing AAV vectors. In one embodiment, the present invention provides formulations useful in the mediation of cell attachment to, or infection by, AAV. The formulation contains an AAV vector along with a soluble receptor-like molecule or artificial receptor of the present invention, preferably in a physiologically or pharmaceutically acceptable carrier. The AAV vector in such a formulation may optionally contain mutations in the binding site for the receptor that enhance binding to the receptor. A second embodiment is a formulation useful in preventing or inhibiting binding of the AAV vector to a cell comprising an AAV vector along with a molecule that blocks binding of the vector to the natural receptor. This formulation will aid in specific targeting of AAV vectors. The AAV vector in such a formulation may optionally contain mutations in the binding site for the receptor that inhibit binding to the receptor. Formulations of the present invention may optionally contain certain additives such as stabilizers or protease inhibitors known to one skilled in the art. Furthermore, AAV vectors provided in formulations of the present invention may optionally comprise heterologous genes that are to be delivered into a target cell for the purpose of expressing the heterologous gene in the cell, e.g., for gene therapy.
A further aspect of the present invention is a kit for mediating AAV attachment to, and infection of, a cell. Such a kit will comprise an AAV vector along with at least one compound that mediates AAV attachment to and infection of a cell, preferably packaged together in a container with written instructions for using the kit.
A further aspect of the present invention is a kit for screening cell samples for permissiveness to AAV infection. Such a kit will comprise a first reagent that binds to the AAV receptor, such as an antibody to the receptor, along with a second reagent for detecting binding between the AAV receptor and the first reagent. The reagent that specifically binds to the AAV receptor and the detecting reagent are preferably packaged in a single container along with written instructions for using the components of the kit to determine if a cell sample is permissive for AAV attachment and infection.
A further aspect of the present invention is a method of enhancing the delivery and transduction of a heterologous gene into a cell, wherein the heterologous gene is delivered into a cell by an AAV vector. In such a method, the heterologous gene is carried by an AAV vector produced using methods known to those skilled in the art. In the present invention, the AAV vector is contacted with the target cell, wherein the target cell is exposed to a soluble receptor-like molecule or artificial receptor of the present invention. In an alternative embodiment, the AAV vector carrying the heterologous gene is contacted with the cell simultaneously with the soluble receptor like molecule.
The discovery that xcex1vxcex25 integrin serves as a co-receptor to facilitate infection by AAV is related to a further aspect of the invention, which is a method of facilitating or enhancing infection of AAV into a cell by treating the cell with a compound that induces or enhances the expression of integrin (preferably, xcex1vxcex25 integrin) on the surface of the cell. Illustrative compounds for upregulating cell surface integrin include cytokines (including interleukins, e.g., IL-1b), hematopoietic growth factors, (e.g., granulocyte-macrophage colony stimulating growth factor and macrophage colony stimulating growth factor), and phytohemagglutinin. As a further aspect, also provided are methods of screening a cell or cell sample for permissiveness to infection by AAV by detecting the presence or absence (or alternatively, the abundance) of integrin (preferably, xcex1vxcex25 integrin) expression on the surface of the cell(s). Also provided, as a further aspect, is a kit for determining if a cell is permissive for infection by AAV, where the kit provides reagents for detecting the presence or absence (or alternatively, abundance) of integrin (preferably, xcex1vxcex25 integrin) on the cell surface.
These and other aspects of the present invention will be set forth in more detail in the description of the invention below.